F = 6.34, p = 0.0144) than the mean levels in the control group (3.2 ± 1.0 pg/mL). The levels of Big ET-1 in patients with essential HTN (0.377 ± 0.1 pmol/L) were similar to those in the control group (0.378 ± 0.07 pmol/L) and did not differ significantly . These data suggest that ET-1, but not Big ET-1, may play an important role in the pathogenesis of primary HTN.The role of endothelin-1 (ET-1) in the pathogenesis of hypertension (HTN) is not clearly established. There is evidence that its circulating levels are elevated in some forms of experimental and human HTN, but this was not a consistent finding. Based on these controversial data, we tested serum levels of ET-1 and Big ET-1 (the precursor of ET-1) in patients with essential HTN, comparing the results with those of healthy normotensive controls. The levels of ET-1 and Big ET-1 were measured by ELISA. Our results in patients with essential HTN showed that the mean levels of ET-1 (5.01 ± 2.1 pg/mL) were significantly higher ( Hypertension (HTN) is one of the most prevalent diseases worldwide and is among the most important risk factors for cardiovascular and cerebrovascular complications . It is cET-1 is a vasoactive peptide identified in 1988 by Yanagisawa and colleagues from the supernatant of porcine aortic endothelial cells (ECs). It is composed of 21 amino acids and two intrachain disulfide linkages in the molecule . In the The role of ET-1 in the pathogenesis of HTN is not clearly established. It is assumed that under physiological conditions, the vasodilating action of ET-1 may predominate, whereas under pathophysiological conditions, ET-1 may behave as a vasoconstrictor and play a role in the pathophysiology of HTN . There in = 20; mean age 47.9 ± 11.3 years) and hypertensive group, patients who were treated at the University Hospital “Dr. Georgi Stranski”—Pleven, Bulgaria . The serum samples of patients and controls were taken from 17 to 21 February 2015. The clinical characteristics of the groups are shown in The study population consisted of 80 persons: normotensive control group ;Willingness to voluntarily participate in the study and sign an informed consent form.Male or female aged ≤35 years and ≥80 years;Secondary HTN and pulmonary HTN;Chronic renal disease, heart failure, liver dysfunction, and malignant tumor;Inability or unwillingness to participate in the study or to sign an informed consent form.Healthy men or women aged ≥35 years and ≤80 years;Individuals with normal BP (SBP 120–129 mmHg and DBP 80–84 mmHg);Willingness to voluntarily participate in the study and sign an informed consent form.BP was measured using a standard cuff mercury sphygmomanometer on the left arm in a sitting position, after 5–10 min rest. The assessment of the arterial HTN was made according to the 2018 ESC/ESH Guidelines for the management of arterial HTN . Normal To measure the concentrations of ET-1 and Big ET-1, blood was drawn into serum tubes and was centrifuged at 2500 rpm for 10 min. Until immunological assay was performed, the serums were stored at −70 °C. The concentrations were measured by ELISA method using commercially available kits.To measure ET-1 concentrations, an ELISA kit from Biomedica Medizinprodukte GmbH & Co. KG, Vienna, Austria (Cat. No. BI-20052) was used according to the manufacturer’s instructions. Serum samples were assayed at 450 nm on an automatic micro-ELISA plate reader .To measure Big ET-1 concentrations, an ELISA kit from Biomedica Medizinprodukte GmbH & Co. KG, Vienna, Austria (Cat. No. BI-20082H) was used according to the manufacturer’s instructions. Serum samples were assayed at 450 nm on an automatic micro-ELISA plate reader .F-test (ANOVA). Values of p < 0.05 were considered statistically significant.Statistical analyses were performed using Statgraphics Centurion XVI software . The data were expressed as mean ± standard deviation (SD). The differences between the groups were assessed by Fisher’s F = 6.34, p = 0.0144) than the mean levels in the control group (3.2 ± 1.0 pg/mL)  . These dF = 0.00, p = 0.9531)  were similar to those in the control group (0.378 ± 0.07 pmol/L) and did not differ significantly ( 0.9531) . These dAccording to the results discussed above, probably ET-1, but not Big ET-1, may play an important role in the development of essential HTN. The controversial and not always consistent results regarding elevated ET-1 levels in patients with primary HTN are probably related to two main reasons. The first is that its elimination from the blood is too fast  . The secOne of the first comparisons of ET-1 concentrations in people with HTN was made between pheochromocytoma patients and healthy controls. Higher levels of ET-1 were observed in patients with pheochromocytoma. In this report, the authors note that HTN in patients with pheochromocytoma is mainly catecholamine-dependent, but may be secondarily ET-1-dependent . These dThe results of our study in patients with essential HTN showed significantly higher serum concentrations of ET-1 compared to normotensive controls . ElevateIn contrast to the concentrations of ET-1, which were significantly higher in patients with HTN, the concentrations of the precursor Big ET-1 did not differ significantly from those of controls . ProbablIn addition to changes in ET-1 levels, endothelial dysfunction at the onset of HTN has also been documented through the use of other circulating biomarkers, such as asymmetric dimethylarginine (ADMA), oxidized LDL, and endothelial microvesicles. Over the last years, ADMA, the endogenous inhibitor of endothelial nitric oxide synthase (NOS3), has emerged as a cardiovascular molecule reflecting endothelial dysfunction and the vascular changes observed in essential HTN ,41.In summary, our results indicate that serum ET-1 levels are elevated in patients with essential HTN and could play an important role in the development of the hypertensive process. Increased production of ET-1 in the vascular wall may promote oxidative stress and low-grade inflammation, with the development of endothelial dysfunction and increased vasoconstrictor activity. Increased ET-1 production can contribute to arterial aging and the development of atherosclerotic changes, which are associated with increased arterial stiffness and manifestation of isolated systolic HTN. In addition, ET-1 is involved in the complex regulation of BP through synergistic interactions with angiotensin II, regulates the production of catecholamines and sympathetic activity, affects renal hemodynamics and water–salt balance, and regulates baroreceptor activity and myocardial contractility. All these effects of ET-1 in the conditions of its increased production can lead to a permanent increase in BP and the development of HTN . The lev


Coronavirus disease 2019 (COVID-19) has affected millions of people worldwide. The infection is mostly spread through the inhalation of infected droplets. Saudi Arabia is a vast country having different climatic conditions.2) were retrieved from official sites. The data was analyzed to determine the effect of these factors on the spread of COVID-19. SPSS IBM 25 software was used to conduct the analysis and p < 0.05 was considered to indicate the significance of the results.The study evaluated the influence of environmental factors on the spread of COVID-19. Six zones (A to F) were classified depending on the climatic conditions. The study was conducted by retrospective analysis of COVID-19 records from the ministry of health between the months of September 2020 and August 2021. The environmental data such as average temperature (°C), humidity (%), wind speed (m/s) and sun exposure (kwh/mp < 0.05) relationship between the environmental factors and the spread of COVID-19. The data suggested that during summer condition when the weather is hot, less humid, and steady wind flow with lots of sun exposure, the COVID-19 infection rate got augmented in Saudi Arabia. Poor ventilation and closed-door habitats in an air-conditioned atmosphere during this period could have played a role in human transmission. More research on air quality, population mobility and diseased condition is essential, so that precise proactive measures can be designed to limit the spread of infection in specific climatic seasons.According to the findings, the rate of infection was greater between April and July 2021. Six climatic zones experienced high temperatures, little humidity, consistent wind flow, and intense sun exposure throughout this time. The correlation study revealed a significant ( Millions of people worldwide were affected by Coronavirus Disease 2019 (COVID-19), which was caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Almost every nation has suffered both economic and human loss due to the spread of the virus. The World Health Organization declared the infection pandemic and recommended several measures to prevent as well as treat the infection . The infThe major route for person-to-person transmission of COVID-19 is through inhalation of infected air. Infected people’s coughs, sneezes, and speech spread the virus and are thought to be the primary causes of infection spread in the community . The firThe incubation time for presenting the clinical symptoms is reported to be 2–5 days, and practically during this duration, the infected person can transmit the virus to others. The average time between the onset of symptoms and the onset of disease complications is 14 days . There aA study conducted in Saudi Arabia suggested that cold weather supports the survival and multiplication of SARS-CoV-2. Their findings indicated that a decrease in temperature, humidity, and wind speed contributed to elevation of COVID-19 cases in Saudi Arabia . HoweverSaudi Arabia is a vast country spread between the Red Sea and the Persian Gulf. It consists of deserts, mountains, valleys, forests, and the ocean. The weather and climatic conditions in different regions of Saudi Arabia varies extremely. In some places the weather is cold and in some it is extremely hot . The intAnalyzing the role played by external factors such as weather conditions in the spread of COVID-19 could assist in designing proactive measures for effective control of infection. Hence, this study was planned to investigate the influence of these factors on the infection rate in different climatic zones of Saudi Arabia.Saudi Arabia is a vast country having distinct climatic conditions in different regions and is mostly due to variation in land elevation. In some places, it is at 0 m above sea level and in some places as high as 3,000 m. The variation in the elevation of land above sea level has contributed in extreme climatic conditions . After sZone-A (Hot-dry with maritime):  Regions included are Dammam, Dahran, Khobar and Hofuf.Zone-B (Cold-dry with desert): Regions included are Tabuk, Jowf, Arar and Hail.Zone-C (Hot-dry with desert):  Regions included are Riyadh, Buraidah, Madinah and parts of Makkah.Zone-D (Hot-dry with maritime desert): Regions included are Jeddah, Jizan, Yanbu and parts of Makkah.Zone-E : Regions included are Taif, Abha, Najran and Khamis mushaitZone-F (Hot-dry desert with empty quarter): Regions included are Abqaiq, Qatif, parts of Ahsa and parts of Hafar Albatein.https://Covid19.cdc.gov.sa/daily-updates/). The number of confirmed cases reported daily for each zone was recorded in an excel sheet and then segregated into respective months. A one-year record of the total number of COVID-19 cases detected in different climatic zones of the country was recorded between September 2020 and August 2021. The data on the weather conditions in different regions of the country was collected from the official website ‘National center for meteorology, KSA’ . The information such as minimum, maximum, and average recorded temperature (°C), humidity (%), wind speed (m/s) and total sun exposure (kwh/m2) was recorded . Detailed information about the number of positive cases detected in different parts of the country was retrieved from the official website of the Saudi Arabian Ministry of Health    and repr •Mean temperatureC)(° –the average temperature measured every month of the climatic zones is represented as  •Average humidity (%) –the mean humidity measured every month in different regions of the climatic zones is represented as  •Average wind speed (m/s) –the mean value of wind speed recorded every month in different climatic zones is represented as  •2)Total sun exposure  was observed to have the maximum infection rate compared to other zones. The decline continued until December 2020 and January 2021, when the infection rate was found to be at its lowest. From February 2021, the infection rate progressively increased in all zones. Upon comparison, Zone-C (cold dry with desert) recorded the rate of infection, followed by Zone-D (hot-dry with maritime desert) and Zone-A (hot-dry maritime). The peak infection rate recorded for each zone is as follows; Zone-A (hot-dry with maritime) = 10.72 (July 2021), Zone-B (cold-dry with desert) = 8.75 (May 2021), Zone-C (hot dry with desert) = 14.19 (May 2021), Zone-D (hot dry with maritime desert) = 13.28 (May 2021), Zone-E  = 9.06  and Zone-F (hot dry desert with empty quarter) = 9.75 . The decline from the peak rate was uniform for all zones except Zone B (cold-dry with desert) and Zone-C (hot dry with desert), where a fluctuation in the infection rate was observed in June 2021, and July 2021. Further, all the climatic zones showed a lower infection rate in the month of August 2021 .• The data on the mean recorded temperature in different climatic zones in a year is represented in • The record of humidity in different climatic zones of Saudi Arabia indicates that in the month of September 2020 the highest percentage of humidity was observed in the Zone-D (hot dry with maritime desert), while other zones recorded below 40%. The level of humidity progressively increased from September 2020 in all the zones, except in Zone-D (hot dry with maritime desert) where it showed slow decline. The highest level of humidity for Zone-A (hot dry with maritime) was recorded in January, 2021 (59%), Zone-B (cold dry with desert) in January, 2021 (57%), Zone-C (hot dry with desert) in December, 2020 (51%), Zone-D (hot dry with maritime desert) in September, 2021 (67%), Zone-E  in December, 2020 and January, 2021 (60%) and Zone-F (hot dry desert with empty quarter) in January, 2021 (54%). After reaching peak in January 2021, the percentage of humidity started to decline progressively in all zones. The lowest humidity level was recorded for Zone-C (hot dry with desert) (12%) in January 2021. The record of Zone-D (hot dry with maritime desert) indicated less fluctuation and remained steady in all months .• The data for wind speed in different climatic regions is represented in 2), Zone-B (cold dry with desert) , Zone-C (hot dry with desert) , Zone-D (hot dry with maritime desert) , Zone-E   and Zone-F (hot dry with empty quarter) .• The total sun exposure for the different climatic zones can be found in p < 0.05) association between the total number of COVID-19 cases in the month of April 2021 and the environmental variables such as temperature and humidity. In the month of May 2021, the correlation was found to be significant (p < 0.05) between COVID-19—temperature and wind speed was found. The analysis for COVID-19 data in the months of June 2021 and July 2021 suggested a significant association (p < 0.05) between all the environmental factors such as temperature, humidity, wind speed and sun exposure on COVID-19 spread  and positive correlation (Rho = 0.118) for Zone-E  and, negative and significant correlation  for Zone-F (hot dry desert with empty quarter). Other zones although suggested both positive and negative correlations but were found to be non-significant. The data for humidity parameter suggested a significant and positive correlation for Zone-A (hot dry with maritime)  and Zone-D (hot dry with maritime desert) . However, data of other zones did not suggested similarity in correlation. The wind speed data analysis suggested a positive and significant  correlation for only Zone-C (hot dry with desert), while other zones were found to be insignificantly correlated. Analysis of the data recorded for total sun exposure in different climatic zones revealed a negative and significant association for Zone-B (cold dry with desert)  and Zone-E  , whereas other climatic zones indicated non-significant correlation.• Saudi Arabia is a large country and has an area of 2.3 million square kilometers. The climatic condition of the country varies from region to region and is mainly due to land elevations that range from 0 to 3000 m above the average sea level. According to climatic conditions, the regions of the country are scientifically classified into six zones . The wea2 annual sun exposure (p < 0.01) positively influenced the spread of COVID-19  started to show a progressive decline from the month of September 2020 and reached its lowest number in December 2020. During this month, the temperature showed a decline, the humidity increased and wind speed slightly fluctuated  to 5. Anexposure  and haveCOVID-19 . These fCOVID-19 . StudiesCOVID-19 .p < 0.05) association between sun exposure and COVID-19 spread (2)  indicated the lowest COVID-19 infection rate in December 2020 and the corresponding temperature recorded was found to be the lowest (12.1 °C), humidity increased to 53%, and wind speed was steady. The highest infection was observed in July 2021, and the temperature in this month increased to 33.4 °C, with a reduced level of humidity (15%) and moderate wind speed (2.6 m/s)  to 5. Th9 spread . This zoread (2) . Being iread (2) . Previouread (2) . Inadequread (2) .2 (p < 0.05) on COVID-19 (In Zone-C (hot dry with desert), the lowest COVID-19 infection rate was recorded in December 2020, and the temperature was found to be reduced to 15.4 °C, with increased humidity (51%) and steady wind speed (2.1 m/s). The highest recorded infection was in June 2021. The temperature in this month increased to 34.7 °C, with reduced humidity (12%) and slightly enhanced wind speed (2.6 m/s)  to 5. Th2  and rainCOVID-19 . As the COVID-19 . In addiCOVID-19 . FurtherCOVID-19 . PolluteCOVID-19 . TogetheCOVID-19 . SimilarCOVID-19 .2 (p < 0.05) correlation between humidity and COVID-19 (The climatic Zone-D (hot dry with maritime desert) recorded the highest and lowest COVID-19 infection rate in the month of January 2021 and May 2021, respectively. The corresponding temperature was found to be slightly reduced 26.5 °C) and increased (32.1 °C), while humidity decreased (60%) and then became constant (56%) with steady wind speed during these periods  to 5. Th °C and i2 . The assCOVID-19 . As the COVID-19 .2) (p < 0.05), and a negative and significant association of sun exposure  with COVID-19 spread  suggested that the lowest recorded COVID-19 infection rate was in December 2021, and the corresponding weather conditions were a drop in temperature 16.5 °C), increased humidity (61%) and steady wind flow (2 m/s). The highest rate of infection was observed in July 2021, with an increased temperature (29.1 °C), decreased humidity (25%) and enhanced wind speed (2.8 m/s)  to 5. Th °C, incr2) . The cor9 spread . Earlier9 spread . The wea9 spread . Being a9 spread .2 (p < 0.05) for the COVID-19 (The data obtained from Zone-F (hot dry desert with empty quarter) indicated the lowest and highest COVID-19 infection rate in January 2021 and June 2021, respectively. Corresponding weather data was temperature decreased in December 2020 (16.3 °C) and increased in June, 2021 (34.8 °C), humidity increased 54%) and then decreased (38%) and wind speed was 2.1 m/s in December, 2020 and 3.1 m/s in January, 2021  to 5. Th4% and th2 . The corCOVID-19 . The regCOVID-19 .Overall, the data from the present study indicated that the rate of infection increased in all the climatic zones when the weather becomes hot, less humid, and steady wind flow. Temperature is one of the important characteristics that is reported to directly influence the immune activity of the host system . ExtremeThe major portion of Saudi Arabia is reported to experience a peak temperature between the months of May and August every year . FurtherAccording to the literature, the amount of sun exposure is directly responsible for the antimicrobial activity. The powerful rays from the sun, such as ultraviolet, can cause damage to microbial cell walls, decreasing the chance of infection . The datThe decline in the COVID-19 infection from the month of August 2021 could be related to several factors. Saudi Arabia is known to have implemented one of the strictest precautionary measures such as wearing of face marks, social distancing, preventing crowding in public places and avoiding social gatherings . The couThe observations of the present study have provided a preliminary data on the spread of COVID-19 in six climatic zones of Saudi Arabia. Although, biological characteristics of the SARS-CoV-2 does not change between regions but its transmission and infection rate are reported to get influenced by the environmental and population-based factors . In the The current research examined the environmental factors that would have influenced the spread of COVID-19 in Saudi Arabia’s six climatic zones. According to the data, humid conditions in maritime zones, high temperature in hot-dry zones, inadequate sun exposure in cold-dry zones and tourist activities in subtropical zones might have influenced the COVID-19 infection rate. All the zones experienced peak infection rate between the months April 2021 and July 2021 that corresponds to summer condition in the country. Close-door activities in hot-dry places, insufficient ventilation in humid regions, inadequate sun exposure in cold-dry areas and movements of travelers in the subtropical Mediterranean quarters might have augmented the transmission rate of COVID-19 infection. The healthcare authorities and public can have a look on these aspects and can design suitable strategies to avoid close-door activities, improve the ventilation, increase the sun exposure and restrict the vocational events to prevent the next wave of pandemic in their regions of habitats. In the absence of specific link between other factors such as role of air-quality, diseased states and population mobility, more research is suggested in this direction to determine their influence on the rate of COVID-19 infection.10.7717/peerj.12732/supp-1Supplemental Information 1Click here for additional data file.


Aronia melanocarpa (AM) fruits are a rich source of phenolic compounds possessing lipid-modulating effects. The present study investigated the effect of 3-month supplementation of AM-based functional beverages on the lipid profile of healthy aging rats. Male Wistar rats (n = 40) were separated into five groups: (YC) young controls (2-month-old); (AC) adult controls (13-month-old); (A) adult animals supplemented with pure AM extract; (A + P) adult animals supplemented with pectin-enriched (1%) AM extract; (A + H) adult animals supplemented with AM extract enriched with a herbal mixture. Total cholesterol (TC), triglycerides (TG), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C) and atherogenic indices were investigated at the end of the study. Adult controls demonstrated age-related dyslipidemia resulting in decreased HDL-C, and increased TG and TC/HDL index. The supplemented groups showed a significant increase in HDL-C levels: A + P (1.49 mmol/L) and A + H (1.61 mmol/L), respectively, vs. AC (1.09 mmol/L), p < 0.05. The TC/HDL-C and LDL-C/HDL-C indices were decreased in the A + P and A + H groups in comparison to the AC group (p < 0.05). These results indicate that supplementation with polyphenol-rich AM beverages can successfully alter HDL-C levels and this effect is further potentiated by pectin and herbs.Plant-based foods rich in phenolic phytochemicals are among the promising strategies to counteract age-related changes in lipid profile.  Age and sex are physiological factors that strongly affect plasma lipid levels in a number of species. Dyslipidemia is a metabolic alteration characterized by elevated fasting blood levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and decreased levels of high-density lipoprotein cholesterol (HDL-C). Dyslipidemia is known to be a risk factor for developing insulin resistance, endothelial dysfunction, hypertension and, most of all, cardiovascular disease (CVD) . FurtherLow HDL-C levels present an unresolved treatment problem. It has been found that lifestyle changes increase HDL-C, and many of the mechanisms underlying its occurrence have been revealed. The latter include physical activity—5–10% ; cessation of smoking—5–10% ; weight loss—5–20% ; moderate alcohol consumption—5–15% ; Mediterranean diet/unsaturated fatty acids—0–5% (increased atheroprotective lipoproteins). The only group of drugs that modulate HDL-C levels with a proven clinical benefit in lowering cardiovascular incidents is HMG-coenzyme A reductase inhibitors (statins). Among all statins, only two are of practical significance—Rosuvastatin (increases HDL-C up to 10%) and Pitavastatin (increases HDL-C up to 29%). Increasing HDL-C independently with the use of four new drugs has been shown to increase cardiovascular and total mortality. Statin intolerance is a major clinical problem, including myalgia, myositis, hepatotoxicity, statin-dependent diabetes mellitus and rhabdomyolysis. Many of the mechanisms underlying its occurrence have been studied. It is the Achilles’ heel of statin therapy, frequently resulting in the discontinuation of statins. According to the latest evidence, the established statin intolerance is close to 3–5% [Aronia melanocarpa (AM), are among the richest sources of polyphenols in the plant kingdom. Polyphenols, especially antocyanins and proanthocyanidins, are the main group of biologically active components in AM berries [Plant-based foods rich in phenolic phytochemicals are among the promising strategies counteracting age-related changes in lipid profile ,7,8. Pol berries ,10. They berries ,11. The  berries ,13,14. I berries .Research has found lipid-regulating properties in AM; however, most studies use different disease models, and evidence of the effect of black chokeberry juice or extracts in models of healthy aging animals is scarce. In a previous study of ours, the supplementation of aging rats with pure black chokeberry juice ad libitum, at a dose of 64 mL/kg per day, improved their lipid profile by lowering TC and LDL, as compared to the adult controls. However, high content of fructose, glucose and sorbitol in the juice resulted in a significant increase in the body weight of the animals under study. In order to avoid such an effect and look for the possibility of applying AM products in functional nutrition, we decided to conduct a new experiment with a modified fixed dose of 10 mL/kg, and variants including enrichment with pectin and herbal extracts .Sambucus nigra) and rose hips (Rosa canina) are known to be rich in polyphenolic compounds belonging to different classes and exhibit a biological activity associated with lipid metabolism [A number of fruits  contain pectin, which has lipid-lowering properties. Pectin normalized body weight in rats following a lipid-rich diet, along with dyslipidemia, hyperglycemia, hyperinsulinemia, metabolic endotoxemia and systemic inflammation . Aside ftabolism ,21. At ptabolism ,23. Regutabolism . TherefoAronia melanocarpa berries were supplied by the licensed farmer Todor Petkov  in the stage of full maturity in August 2017. Fresh fruits were packed in polyethylene bags, frozen immediately and stored at −18 °C until the time of juice production. To prepare the beverage, 5 kg of frozen fruit were defrosted at room temperature and homogenized in a laboratory blender.Rosa canina (rose hip) fruits were harvested from the Rhodope Mountains in the autumn of 2016, frozen at −18 °C and freeze-dried for 96 h in Alpha 1–4 LDplus laboratory freeze dryer. After that, dried rose hips were deseeded and stored in paper bags at room temperature until the time of production of the functional beverage.Sambucus nigra) flowers were purchased from a local pharmacy in Plovdiv, Bulgaria, ground to a fine powder in a laboratory mill and stored in paper bags prior to preparation of the extract.Dried elder (g) in a Megafuge 1.0R bench top centrifuge .To prepare the aronia extract, 400 g of fruit homogenate were mixed with 600 mL ultrapure water, transferred to a brown-glass bottle and incubated in a thermostatic water bath shaker  for 1 h, at 60 °C. After this, the mixture was filtered through a cheese cloth and the liquid phase was centrifuged  in a Megafuge 1.0R bench top centrifuge . For the preparation of the aronia beverage with herbs, 360 g of aronia homogenate were mixed with 40 g rosehips husks (without seeds), 20 g elder flower powder and 600 mL ultrapure water. The mixture was transferred to a brown-glass bottle and incubated in a thermostatic water bath shaker  for 1 h, at 60 °C. After this, the mixture was filtered through a cheese cloth and the liquid phase was centrifuged , using the Folin–Ciocalteu reagent. Gallic acid was used for the calibration curve and the results were expressed as gallic acid equivalents (GAE) per liter of beverage.ORAC was measured by the method of Ou et al. (2001). ORAC analyses were carried out on a FLUOstar OPTIMA plate reader  with an excitation wavelength of 485 nm and emission wavelength of 520 nm. The results were expressed as μmol of Trolox equivalents per liter (μmol TE/L).Polyphenol content and ORAC antioxidant activity of the AM-based functional beverages under study are shown in n = 8). Eight two-month-old rats and eight 13-month-old rats were designated as young controls (YC) and adult controls (AC), respectively. Three groups were given orally polyphenol-rich drinks diluted with drinking water in the course of 3 months, the supplemented animals being the same age as the adult controls. The groups were as follows: (A) 100% AM extract in a dose of 10 mL/kg; (A + P)—AM extract enriched with 1% pectin in a dose of 10 mL/kg; (A + H)—AM extract enriched with a herbal mixture (rosehip fruit and elderflowers) in a dose of 10 mL/kg. The rats were kept in compliance with all the experimental procedures recommended by the European Commission for protection and welfare of laboratory animals. The experimental protocol was approved by the Committee on Ethical Treatment of Animals of the Bulgarian Agency for Food Safety (No. 193/2018). All animals received humane care in compliance with the “Principles of laboratory animal care” formulated by the National Society for Medical Research and the “Guide for the care and use of laboratory animals” prepared by the National Institute of Health .The present study included 40 male Wistar rats. The animals were bred in the vivarium of the Medical University, Plovdiv under standard laboratory conditions . Taking into account the hormonal influences in females, we used only male rats. The animals were divided into 5 groups (2 (cm2)) and heart weight index (heart weight (g)/body weight (g) × 100) were calculated.Body measurements were taken at the end of the experiment—body weight (g), body length (cm) and heart weight (g). Body mass index (body weight (g)/lengthg for 10 min. Serum triglycerides  and total cholesterol  were determined by the enzyme-colorimetric method. Serum levels of high-density lipoprotein cholesterol  and low-density lipoprotein cholesterol  were determined by the direct enzyme-colorimetric method . The atherogenic indices were determined as TC-C/HDL-C ratio and LDL-C/HDL-C ratio.At the end of the experiment, all animals were anesthetized with i.m. Ketamin (90 mg∙kg) + Xilazine (10 mg∙kg) and then decapitated. Immediately after decapitation, blood was collected, placed in a centrifuge tube and allowed to clot, so that serum could be obtained. The latter was removed by centrifugation at 1400× t-test, independent sample t-test and one-way ANOVA followed by Tukey’s test were used for the parametric analysis at a normal distribution level. Wilcoxon signed rank test and Mann–Whitney U tests were used for the non-parametric analysis. p < 0.05 was considered as statistically significant.The data obtained were processed statistically using SPSS 21. The results are presented as mean ± standard error of mean (SEM). Paired sample p < 0.05), which is a natural occurrence in the aging process. Body weight and body mass index in the adult-supplemented animals did not change significantly, as compared to the adult controls , as compared to the young controls . For example, YC (0.515 g) vs. AC (1.120 g), (p < 0.05), YC (0.515 g) vs. A (1.160 g), (p < 0.05). No significant difference was found in these parameters when comparing individuals from the supplemented adult groups , as compared to the young group  and HDL-C (1.55 mmol·L−1) of YC were significantly different (p < 0.05) in comparison to the values found in AC—1.25 mmol·L−1 and 1.09 mmol·L−1, respectively—resulting in a significantly lower (p < 0.05) TC/HDL index in YC (1.29), as compared to AC (1.71) (−1 vs. 1.85 mmol·L−1) and LDL-cholesterol (0.34 mmol/L vs. 0.35 mmol/L) levels in the young and adult controls  A. Howevecontrols A–D.p < 0.05), whereas the atherogenic TC/HDL index in the A + P and A + H groups  was significantly lower than the one found in AC (1.71), (p < 0.05)  were higher than that of the AC group (1.09 mmol/L), ( < 0.05) A–D. In a < 0.05) A and 4B.−1 does not lead to significant body weight alterations. These results showed that supplementation with functional beverages did not have an effect on the body mass index—the criterion for normal body structure. The significant differences between the weight index of the internal organs of the animals from the young and adult groups (supplemented and not supplemented) are an indication of the age-related atrophy of these organs. The absence of significant differences between the weight indices of the supplemented animals and the adult controls suggests that the weight of an organ changes in correlation to the overall body weight as a result of supplementation, which has been described by other authors [From the results, it is evident that body weight alterations in the animals increase with age—a fact that has been found by other authors as well. Body weight and body mass index change significantly with the aging process, which is due to an increase in the body adipose tissue, on the one hand, and to changes in the lean mass, on the other ,26,27. I authors . FurtherThe occurrence of age-related dyslipidemia in healthy middle-aged rats was confirmed by the significant differences in the lipid profile, as compared to the young group  A–D. TheThe current study involved healthy normally aging animals and followed the natural development of age-related dyslipidemia. Studies in animals and humans have shown that AM berries effectively modulate lipid metabolism ,32,33. IResearch on the mechanisms of AM lipid-lowering effect has found that black chokeberry extracts exert an effect on the expression of genes regulating cholesterol synthesis, binding and elimination in a dose-dependent manner in humans. The data obtained by Kim et al. suggest that the hypolipidemic effects of AM extract are likely to be due to an increased apical efflux of LDL-derived cholesterol and to decreased chylomicron formation in the intestines .O-β-glucoside to mice by an oral gavage (50 mg/kg body weight) has found that the substance markedly increased serum HDL-C and apo A-1 level, as well as reduced atherosclerosis [Fragaria nilgerrensis Schlecht. and Centella asiatica (L.) Urban in combination significantly decreased the levels of TG, TC, LDL-C, apolipoprotein B and hepatic malondialdehyde, but increased serum HDL-C, apolipoprotein A1 and hepatic superoxide dismutase [Polygonum cuspidatum Sieb., a widely used herb in traditional Chinese medicine, has been found to influence the lipid and glucose metabolism in mice, significantly lowering serum TG and LDL-C levels, simultaneously increasing HDL-C levels, as compared to the control mice [A particularly interesting finding of the study is the increase in the HDL-C levels in the groups supplemented with combined beverages—pectin-enriched AM and AM enriched with a herbal mixture. Such an effect on HDL-C is in the focus of attention of present-day research. In epidemiological studies, HDL-C is considered to be a risk marker for cardiovascular disease, so inducing elevation of HDL-C levels is a controversial issue. Since many of the drugs influencing HDL-C levels either exhibit serious side effects, or do not lower the risk of developing cardiovascular diseases, there is an active search for alternative means other than medications . An expeclerosis . On the clerosis . In an eismutase . A comporol mice . In our rol mice . Studyinrol mice . A 90-darol mice .The results from the present research showed that the administration of AM juice in combination with a herbal mixture have a potentiating effect on HDL-C. This is likely to be due to the rich polyphenolic content and the synergism with the other biologically active substances. The elderflowers have a high polyphenolic content, of which phenolic acids, quercetin, kaempferol, catechin, epicatechin and narginin constitute a major part. They can affect disease processes by counteracting oxidative stress, leading to a positive effect on blood pressure and blood sugar levels, stimulating the immune system, increasing plasma enzymatic antioxidant activity and lowering uric acid . Rose hiAronia melanocarpa in combination with pectin or a herbal mixture has the potential to be used not only for prophylaxis, but also as an adjunct functional food in the diet for the purposes of managing dyslipidemia.The optimal balance between “bad” (LDL-C) and “good” (HDL-C) in the atherogenic process is part of the history of medicine. Numerous epidemiologic findings have confirmed the fact that low HDL-C levels are associated with high cardiovascular mortality ,4,43. OnThe results obtained in the present experimental investigation regarding the significant elevation of HDL cholesterol following supplementation with black chokeberry extract require further clinical studies involving patients. Furthermore, such an elevation in HDL-C could have an extensive clinical application under the conditions of the COVID-19 pandemic. There is evidence that HDL-C plays an important part in the normal functioning of the human immune system . Recent Aronia melanocarpa could counteract spontaneous age-related dyslipidemia in healthy adult rats by modulating serum HDL-cholesterol levels. The addition of pectin or herbs further increases Aronia melanocarpa lipid-lowering effect. More specifically, the combination of aronia with rosehip and elderflower extracts had the most pronounced HDL-cholesterol increasing effect. Therefore, the functional beverages studied can be used as a potential means for prophylaxis and as an adjunct functional food in the diet for the purposes of managing dyslipidemia.The results of the present study indicate that nutrition-relevant doses of functional beverages based on


Background: TNF-α has been reported to be closely associated with autoimmune inflammatory diseases. This study aims to investigate the role of TNF-α -308(rs1800629) G/A gene polymorphisms as well as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in predicting the efficacy and safety of TNF inhibitors (TNFi) in patients with ankylosing spondylitis (AS), rheumatoid arthritis (RA), and psoriasis arthritis (PsA).Methods: A total of 515 subjects  were recruited in this study. The accuracy of RT-PCR methods for identifying individual TNF-α -308 genotypes was assessed using sequencing as the gold standard. Baseline NLR and PLR of patients with AS, RA and PsA and healthy controls (HC) were calculated and compared. Meanwhile, differences between responders and non-responders to TNFi treatment as well as between individuals with and without adverse effects (AE) among responders were compared.Results: The RT-PCR method is stable and reliable for TNF-α -308G/A gene polymorphism analysis, independent of sample status. The GG genotype was overwhelmingly represented, with relatively few GA genotype, whilst the AA genotype was not detected in this study. There was no observed association between TNF-α-308G/A polymorphism and susceptibility in AS, RA or PsA patients. Patients with AS, RA, and PsA had a higher NLR, compared to the HC group. Apart from PsA patients, AS and RA patients had a higher PLR, compared to the HC group. NLR was positively correlated with PLR. Furthermore, a lack of response was more frequently observed in AS and RA patients that carrying the GA genotype than the GG genotype. AS and RA patients with AE had higher NLR and PLR, compared with the non-AE group.Conclusion: Our study preliminarily shown that combining TNF-α -308G/A polymorphisms with NLR and PLR can predict the responsiveness and safety of anti-TNF therapy in patients with AS or RA. Ankylosing spondylitis (AS), rheumatoid arthritis (RA) and psoriasis arthritis (PsA) are common autoimmune-mediated inflammatory diseases. The primary clinical feature of AS is the involvement of peripheral joints, which eventually leads to ossification of entheses and loss of joint mobility . AccordiTNF-α promoter are gradually being of interest. The G-to-A transition at position -308 (rs1800629) has been the most extensively investigated. However, the association of SNP at the TNF-α -308G/A with autoimmune diseases such as AS, RA, and PsA has not been fully elucidated.Tumor necrosis factor alpha (TNF-α) is a cytokine that produced by macrophages or monocytes in acute inflammation which involved in a range of cellular signaling events . Serum lIn recent years, there is growing evidence of the value of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in assessing disease activity and prognosis in systemic inflammatory diseases . BesidesTNF-α -308G/A can be an excellent predictor of the responsiveness to TNF-α inhibitors  have been introduced in autoimmune diseases and have shown favorable performance . Howeverhibitors . Similarhibitors . SpecifiTNF-α -308G/A polymorphisms, NLR, and PLR as potential biomarkers in AS, RA, and PsA, as well as the role of TNF-α -308G/A polymorphisms in combination with NLR and PLR in predicting the efficacy and safety of anti-TNF-α therapy.In this study, we focused on the utility of A total of 515 subjects  were recruited from Peking Union Medical College Hospital (PUMCH), Chinese Academy of Medical Sciences between July 2019 and August 2021 in this study. Of these patients, 181 patients were diagnosed with AS according to the Modified New York criteria by the American College of Rheumatology . 144 patThis study was conducted in accordance with the recommendations of the PUMCH. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethics Committee of PUMCH (No. HS2019035).TNF-α -308 (rs1800629) was performed using TNF-α -308G/A gene polymorphisms RT-PCR detection kit . Genotypes were interpreted with the criteria of Genomic DNA was extracted from whole peripheral blood via DNA extraction kits . Genotyping for http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). p < 0.05 was considered statistically significant.All the data were analyzed by Excel 2019 , and SPSS 20.0  software. The concordance between genotyping methods was analyzed by using kappa test. Correlation tests were performed by using the Spearman’s correlation analysis. Differences between groups were assessed using the Wilcoxon or Chi-square test. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using logistic regression analysis. The Hardy-Weinberg equilibrium (HWE) was evaluated by online website (TNF-α -308G/A polymorphisms detection kit (RT-PCR) was used to identify the genotype of each subject. To ensure the reliability of genotype identification for each participant, a subset of subjects  were selected to assess the accuracy of the RT-PCR method, using the sequencing method as the gold standard. As depicted in In this study, the TNF-α -308G/A. The clinical characteristics and baseline laboratory data of all participants were summarized in TNF-α -308G/A genotypes were shown in p = 0.52). Overall, the GG genotype was overwhelmingly represented, with relatively few GA genotype, whilst the AA genotype was not detected in this study. Furthermore, logistic regression analyses revealed that TNF-α -308G/A polymorphisms were not associate with susceptibility of AS, RA, or PsA (p > 0.05).A total of 181 patients with AS, 144 with RA, 48 with PsA and 122 HC were included in the study to examine the polymorphisms of ). We further compared the differences of NLR and PLR among individuals with different genotypes and showed that only in the RA group, patients with the GA genotype had a higher NLR than the GG genotype. . It was intriguing to note that NLR was positively correlated with PLR in the AS , RA , PsA  and HC groups . . Moreover, we observed a positive correlation among NLR, PLR, CRP and ESR in the AS and RA groups. . . Moreov groups. .via the HIS system. As illustrated in In this study, we collected information on medication administration for all patients p < 0.05). For RA patients, non-responders had higher level of NLR and PLR compared to responders, whereas for AS patients this difference was not observed. Therefore, TNF-α -308G/A polymorphisms may be used as a predictor of responsiveness to TNFi.In this study, the AS, RA and PsA groups had 97, 28 and 22 patients who received TNFi such as etanercept or adalimumab. . Most paTNF-α -308G/A polymorphisms with NLR and PLR can predict the responsiveness and safety of anti-TNF therapy in patients with AS or RA.Remarkably, we also observed that most patients carrying the GG genotype showed a superior response to TNFi, but there were still some patients suffering from AE, such as somatic pains, iritis, or tuberculosis, which led them to switch to another TNFi or even abandon anti-TNF-α treatment. Meanwhile, patients carrying the GG genotype have a wide range of values for NLR and PLR. On this basis, we intended to explore whether NLR and PLR could further identify patients exposed to the risk of AE. As shown in TNF-α promoter -308A allele in Crohn’s disease increased TNF production promoting inflammatory activity and were associated with worse responsiveness to anti-TNF-α therapy   , restric-TOF MS) , and hig-TOF MS) . HoweverTNF-α -308 gene is relatively scarce in the Chinese population.Interestingly, the GG genotype was overwhelming (over 90%) in both disease groups and healthy controls, the GA genotype was relatively rare (2.08–6.56%), and the AA genotype was not detected. The frequency of genotype distribution in our study was analogous to two other studies from China . In contTNF-α -308 G/A and AS has been well studied but still controversial. Sode et al. and Hu et al. have reported that heterozygous variant genotypes of TNF-α -308G/A were associated with reduced risk of AS (TNF-α -308G/A (rs1800629) polymorphism were observed between AS patients and healthy individuals . A previous meta-analysis consisting of 2253 psoriasis patients and 1947 controls highlighted that TNF-α -308G/A polymorphism was significantly associated with decreased risk of psoriasis   . PerhapsTNF-α -308G/A polymorphisms and NLR or PLR. Nevertheless, NLR was positively correlated with PLR in AS, RA, PsA patients and in the HC group, revealing a well-defined agreement between the NLR and PLR. When considering the association between NLR, PLR and other laboratory indicators, such as CRP or ESR, higher correlations were observed in patients with AS and RA. These correlations have also been confirmed in previous publications (Generally, the neutrophil count in the blood increases as the inflammatory disease progresses, while the lymphocyte count decreases. There is growing evidence that platelets are activated and play vital roles in the inflammatory response . Thus, Nications , suggestTNF-α -308G/A polymorphisms and responsiveness to TNF-α inhibitors.TNFi can be of significant benefit to patients with AS, especially in cases when conventional treatment has failed, but non-response to its application also raised a concern. On the other hand, TNFi is enormously expensive and carries the risk of adverse effects, thus there is an urgent need for a program to guide the use of the drug based on individual variation. Our survey data showed that 97 patients with AS, 28 patients with RA, and 22 patients with PsA were given treatment with TNFi and most of them exhibited a favorable outcome, while just ten were non-responders. Despite the small size of the non-response cases, the fact that six of the ten cases were of the GA genotype implied that the GA genotype may be a predictor of treatment failure. Our study agreed with the findings of Ma et al., in their study, the GG genotype was more frequent in good responders to TNF-α inhibitors in patients with AS . CertainTNF-α -308G/A polymorphism and responsiveness of TNFi in patients with RA was as well controversial. A meta-analysis conducted by Lee et al. asserted that RA patients carrying the A allele had a worse response to TNF-α inhibitors than those carrying the G allele, but they denied this in a consequent updated meta-analysis (TNF-α -308 G allele shown better responsiveness to TNFi than those with A alleles in Caucasians (TNF-α -308G/A polymorphisms with responsiveness to TNFi in Chinese PsA patients, which of course needs further verification subsequently.The association between analysis . Therefoucasians . UnfortuAnother concern about TNFi is its adverse effects. We have observed that some responders suffered from AE that forced them to change to another TNFi or to give up biological treatments. This situation imposed a heavy financial burden on them and taken a psychological toll on them. Lee et al. reported that in patients with RA, higher baseline NLR predicted poorer persistence of TNFi, including lack of efficacy and adverse events . Since NTNF-α -308G/A polymorphism; other SNPs of TNF-α should be included in future studies.However, there were some limitations in our study. Firstly, our sample size was limited, and larger sample sizes would be required in future studies. Secondly, the A allele was remarkably scarce in our study in the Chinese population, but this was consistent with the relevant published studies across other cohorts. Thirdly, in this study we only examined the TNF-α -308G/A polymorphisms may be used as a potential biological marker to evaluate the responsiveness to TNFi in AS or RA patients. Higher baseline NLR and PLR could identify responders at higher risk of AE. Overall, combining TNF-α -308G/A polymorphisms with NLR and PLR can predict the responsiveness and safety of anti-TNF therapy in patients with AS or RA.Our study preliminarily shown that


Nontuberculous mycobacterial pulmonary disease (NTM-PD) is an uncommon mycobacterial infection characterized by worsening lung function and increased health care resource utilization; however, the overall risk for hospitalization among patients with NTM-PD remains unclear.What is the hospitalization risk among older adults with NTM-PD?A retrospective, nested, case-control study was conducted by using the Medicare claims database. Cases were defined as patients with ≥ 2 NTM-PD claims ≥ 30 days apart between January 1, 2007, and December 31, 2015. The study included individuals aged ≥ 65 years with ≥ 12 months of continuous enrollment in both Parts A and B before the first NTM-PD diagnosis. Cases were matched 1:2 to Medicare beneficiaries without NTM-PD (control subjects) according to age and sex. Hospitalizations following the first NTM-PD claim were compared between case and control subjects by using univariate and multivariate analyses.P < .05) greater among case subjects than control subjects. Case subjects also had a significantly shorter time to hospitalization than control subjects. The increased burden due to hospitalization was reflected in multivariate analysis adjusting for baseline comorbidities. All-cause hospitalization in patients with NTM-PD relative to control subjects was 1.2 times more likely  with a 46% greater hazard .A total of 35,444 case subjects and 65,467 matched control subjects  were identified. Baseline comorbidities, particularly pulmonary comorbidities, were more common in case subjects than in control subjects . All-cause hospitalization was observed in 65.7% of case subjects and 44.9% of control subjects. Unadjusted annual hospitalization rates were significantly (Patients with NTM-PD were significantly more likely to be hospitalized, had greater annualized hospitalization rates, and had shorter time to hospitalization than age- and sex-matched control subjects without NTM-PD. These findings highlight the significantly increased burden of hospitalizations among patients with NTM-PD.  Study Question: What is the hospitalization risk among older adults with NTM-PD?Results: In this nested case-control study of Medicare beneficiaries (2007-2015), both univariate and multivariate analysis adjusting for baseline comorbidities show that patients with NTM-PD had an increased risk of all-cause hospitalization vs control subjects , higher annual hospitalization rates than control subjects , and a significantly greater risk for sooner all-cause hospitalization relative to control subjects at any time during follow-up .Interpretation: Patients with NTM-PD were significantly more likely to be hospitalized, had greater annualized hospitalization rates, and had shorter time to hospitalization than age- and sex-matched control subjects without NTM-PD, highlighting the need for careful medical management of these patients and clinical management strategies that may reduce excess hospitalizations.Nontuberculous mycobacterial pulmonary disease (NTM-PD) is a rare, chronic, progressive, and potentially debilitating condition that has become an increasingly common diagnosis.,,NTM-PD is known to lead to impaired pulmonary function and quality of life and an increased risk of mortality.4In an analysis of national hospital discharge data for the United States from 1998 through 2005, the average annual prevalence of non-AIDS NTM-PD-associated hospitalizations among persons aged 70 to 79 years was 7.6 per 100,000 for men and 9.4 per 100,000 for women, with lower rates observed for younger individuals.International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] 031.0; International Classification of Diseases, Tenth Revision, Clinical Modification [ICD-10-CM] A31.0) that were ≥ 30 days apart.,We conducted a nested case-control study using the 100% US Medicare Parts A and B administrative claims database for January 1, 2007, through December 31, 2015. This population-based database includes beneficiaries in all 50 US states. A case of NTM-PD was defined as ≥ 2 claims with a diagnostic code for NTM-PD . In addition, the Charlson Comorbidity Index (CCI) was calculated based on baseline diseases and health conditions to characterize patients' overall disease burden.Hospitalizations were accrued over the follow-up period, which was defined as the interval between the index date and the end of the study period , the last day prior to enrollment in Medicare Advantage (Part C), or death, whichever was earliest. Over the follow-up period, parameters related to hospitalization were estimated, including the proportion of patients with hospitalizations, the incidence rate of hospitalizations, the risk of hospitalizations, and the time to hospitalization, comparing NTM-PD case subjects vs control subjects for all-cause, COPD-related, pneumonia-related, and other pulmonary disease-related hospitalizations. Causes associated with a hospitalization were identified from the first five diagnostic codes of the inpatient claim (up to 25 diagnostic codes per inpatient claim were available in Medicare claims). When the first five diagnostic codes included more than one cause of interest, the assignment of relevant cause for that hospitalization claim followed the order of COPD, pneumonia, and other pulmonary diseases. Time to hospitalization was measured as the interval between the index date and the date of the first hospitalization during the follow-up period.P < .05.The study includes descriptive analysis of demographic and clinical characteristics of patients with NTM-PD and the age- and sex-matched control subjects, as well as an assessment of hospitalizations accrued over the follow-up period. We calculated the proportions of individuals with hospitalizations, the annualized hospitalization rates, and the time to hospitalizations with both univariate and multivariate analysis. Statistical significance was assessed at 2 test for categorical variables and the two-sided Student t test for continuous variables.Categorical variables are presented as the count and percentage; continuous variables are summarized through mean ± SD. Univariate analysis included the two-sided χPoisson regression was used to estimate the unadjusted annual incidence rates of hospitalizations for NTM-PD case subjects and control subjects as well as the incidence rate ratio (IRR) of case subjects vs control subjects. Rates were estimated separately for persons who survived the follow-up period (follow-up ended due to enrolling in Medicare Advantage or data cutoff) and those who did not (follow-up ended due to death), as more hospitalizations prior to death are often observed independent of the NTM-PD status. Rates and the IRRs for all-cause, COPD-related, pneumonia-related, and other pulmonary disease-related hospitalizations are reported.The relative risk of hospitalization was estimated by using Poisson regression, with the dependent variable defined as a Boolean variable, indicating hospitalization status. Both unadjusted and adjusted relative risks for all-cause hospitalization controlling for baseline comorbidities are reported.Time to hospitalization was described with Kaplan-Meier survival curves stratified according to group (NTM-PD vs control). Censoring events were death, data cutoff, or enrolling in Medicare Advantage. The log-rank test was used to assess the significance of the differences in the time to hospitalization between the two groups. The hazard ratio of NTM-PD relative to control subjects was estimated by using the Cox proportional hazards model with time to all-cause hospitalization as the dependent variable, controlling for baseline clinical factors.Statistical analyses were conducted by using SAS Enterprise Guide .An overview of the study sample construction is provided in 2 test, P < .01).The baseline characteristics of the NTM-PD cohort  and the matched control subjects  are provided in The mean follow-up time was 3.1 ± 2.2 years for patients with NTM-PD and 3.3 ± 2.2 years for control subjects. Proportions of individuals with hospitalizations, annualized hospitalization rates, and time to hospitalizations accrued over the follow-up period are presented in the following sections.P < .0001), indicating that patients with NTM-PD were nearly two times more likely to have a hospitalization than the control subjects. After controlling for baseline comorbidities (P < .0001).All-cause hospitalization was observed in 65.7%  of patients with NTM-PD and 44.9%  of control subjects during follow-up. Of the first all-cause hospitalizations following the index date, 52.5% were pulmonary related  in the NTM-PD group, compared with 19.2% in the control group  . The unabidities , patientP < .001). Hospitalizations related to pulmonary conditions had a significantly higher incidence in patients with NTM-PD relative to control subjects. Similar trends of significantly higher incidence rates associated with NTM-PD were also observed among the individuals who had died at the end of follow-up. For example, the annual incidence rate for all-cause hospitalization was 1.65 in patients with NTM-PD compared with 1.06 in the control group . Regardless of survival status during follow-up, differences in annual rates between the NTM-PD and control groups were approximately threefold or greater for hospitalizations related to COPD, pneumonia, or other pulmonary diseases, with the greatest difference in COPD-related hospitalization admissions.The annual incidence rates of hospitalization were estimated separately based on the survival status at the end of the follow-up period for both the patients with NTM-PD and the control subjects. By the end of the follow-up period, 30.1%  of patients with NTM-PD died, compared with 18.6%  of control subjects; mean follow-up time was 2.3 ± 1.8 years for NTM-PD and 2.6 ± 1.9 years for control subjects. For those who survived at the end of follow-up, the mean follow-up time was 3.4 ± 2.2 years for patients with NTM-PD and 3.5 ± 2.2 years for control subjects. P < .0001).We found that among Medicare beneficiaries aged ≥ 65 years, the risk of all-cause hospitalization in patients with NTM-PD was significantly increased relative to age- and sex-matched control subjects. Overall hospitalization and pulmonary disease-related hospitalization rates were markedly higher for both patients who survived throughout the follow-up period and for those who died during the follow-up period. Pulmonary-related comorbidities were more common in patients with NTM-PD at baseline, accounted for more first all-cause hospitalizations, and likely contributed to the higher rate of all-cause hospitalization. Notably, after adjustment for confounding baseline comorbidities, compared with the control group, patients with NTM-PD had a greater than threefold increased risk of all-cause hospitalization and a 46% higher risk of having all-cause hospitalization sooner than control subjects at any time following the diagnosis of NTM-PD.The increased risk of hospitalization attributable to NTM-PD is supported by other NTM-PD hospitalization studies as well as by studies that examined NTM-associated mortality. Hospitalizations are a marker of disease severity, as people who are hospitalized are more likely to die. One recent autopsy study showed that NTM can cause progressive, fatal disease.The high percentages of patients with COPD (81%) and bronchiectasis (44%) have been reported in analyses of Medicare data from an earlier period (1997-2007),,The risk of hospitalization among people with NTM-PD indicates a very high burden of disease and associated morbidity. Hospitalizations are associated with an increased cost relative to outpatient care, and an improved understanding of the risk of hospitalization attributable to NTM-PD will allow for a better estimation of associated costs.One strength of our analysis is that it provides population-based risk estimates among older adults in the United States. The current analysis was based on Medicare Parts A and B, which represented 69% of total Medicare enrollment in 2015; the majority (83%-84%) of Medicare Parts A and B enrollees were enrollees aged ≥ 65 years in 2010 to 2015.,,The primary limitation of the current study is that the ICD-9-CM/ICD-10-CM codes for NTM-PD have a low sensitivity, resulting in some underdiagnosis. The ICD-9-CM codes for NTM-PD have been evaluated in several studies, and even though their sensitivity is low,Patients with NTM-PD aged ≥ 65 years had statistically significantly more hospitalizations compared with age- and sex-matched control subjects without NTM-PD, highlighting the need for careful medical management of these patients and the need for clinical management strategies that may reduce excess hospitalizations for these patients.


Streptococcus mutans biofilms (S. mutans). Briefly, S. mutans biofilms were formed on saliva-coated hydroxyapatite disks, which were exposed to CS 1, 3, and 6 times per day, respectively. In addition, S. mutans biofilms without CS exposure were considered as the control group. Acidogenicity, dry weight, colony-forming units (CFUs), water-soluble/insoluble extracellular polysaccharides (EPSs), and intracellular polysaccharides (IPSs) were analyzed and confocal laser scanning microscopy (CLSM) images of 74-h-old S. mutans biofilms were obtained. The lowest accumulation of biofilms and EPSs were detected in the 6 times/day CS exposure group compared with those of the control group and other CS exposure groups in 74-h-old S. mutans biofilms. CLSM also revealed the lowest bacterial count (live and dead cells) and EPSs biovolume in the six times/day CS exposure group in 74-h-old S. mutans biofilms. CS exposure inhibited the growth of S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.The increased incidence of dental caries by cigarette smoking (CS) has been widely reported in epidemiological studies, but the relationship between CS and cariogenic biofilm growth has been rarely studied. This study aims to investigate the effects of CS exposure on the growth and virulence of  EPSs contribute to bacterial adhesion on the tooth surface, colonization, and formation of plaque biofilm, which are important cariogenic factors of S. mutans. Besides, acid production through carbohydrates and acid tolerance in low pH is also the major cariogenic factors of S. mutans that result in the loss of local hard tissue and the initiation of the cariogenic process [Dental caries is a dental biofilm-related disease . An impobacteria . Althougbacteria , S. mutal caries , 5. S. m process .CS is an important factor affecting the progress of dental caries. Numerous epidemiological studies around the world have reported a close relationship between smoking and the occurrence of dental caries –11. An aS. mutans biofilms) has been rarely analyzed, which is of great significance to study the influence of CS on the growth of S. mutans biofilms and dynamic bacterial equilibrium for further understanding the relationship between smoking and high caries prevalence. This study aimed to investigate the influence of CS on the growth, virulence (EPSs and acidogenicity), and viability of cariogenic biofilms in vitro by using the S. mutans biofilm model.To the best of the author’s knowledge, the relationship between CS and the growth of cariogenic biofilm  biofilms were formed on saliva-coated hydroxyapatite discs  placed in a vertical position in 24-well plates. Briefly, an adult male (non-smoker) was selected for oral saliva collection. Hydroxyapatite discs were incubated in filter-sterilized (0.22-μm low protein-binding filter) saliva (3 ml/disc) for 1 h at 37°C. For biofilms formation, the saliva-coated hydroxyapatite discs were transferred to a 24-well plate containing brain heart infusion  broth with 1% (w/v) sucrose and S. mutans (5–7×106 colony-forming unit (CFU)/ml) (3 ml/disc). The biofilms were grown at 37°C with 5% CO2 until 74 h (S. mutans biofilms were divided into 4 groups. Experiment group 1 (control group) did not receive CS exposure and are exposed to the air six times per day . Experiment group 2 was exposed one time per day (at 10 a.m.) with CS and five times per day  with air, a total of three times CS exposure in 74-h-old biofilms. Experiment group 3 was exposed three times per day  with CS and three times per day  with air, a total of seven times with CS exposure in 74-h-old biofilms. Experiment group 4 was exposed six times per day  with CS, a total of fifteen times in 74-h-old biofilms. Each exposure time was 5 minutes . The culture medium was changed twice daily (8 a.m. and 6 p.m.) . The hydroxyapatite disks were washed with distilled water three times a day   for the control group and all CS exposure groups. This study is approved by the ethics committee/institutional review board of the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. All experimental protocols were approved by the Department of Preventive Dentistry, School of Dentistry, Institute of Oral Bioscience, Jeonbuk National University. The author confirms that all methods were carried out by relevant guidelines and regulations and informed consent had been obtained from the subject.til 74 h . After 2in vitro method was considered. Considering that S. mutans is a facultative anaerobe, a glass container was designed to allow slight air to enter while CS was inhaled to avoid the effect of complete hypoxia on the growth of S. mutans biofilm. At the time of exposure, the saliva-coated hydroxyapatite discs were taken out from the culture medium, placed in a sterile glass container, and a cigarette was taken using a vacuum machine to simulate cigarette gas in the mouth during smoking, each exposure time was 5 minutes. After exposure, saliva-coated hydroxyapatite discs were returned to the culture medium. This device simulates CS flow has been verified in a previous study , has the highest tar and nicotine content per cigarette of all cigarette brands. We used vacuum machines to provide smoking force.The dry weight and CFUs in the homogenized suspension were analyzed. Briefly, the 74-h-old biofilms on the saliva-coated hydroxyapatite disc were transferred into 2 ml of 0.89% NaCl and sonicated in an ultrasonic bath for 10 min to disperse the biofilms. The dispersed solution was re-sonicated at 7W for 30 s after adding 3 ml of 0.89% NaCl . For the determination of CFUs count, an aliquot (0.1 ml) of the homogenized solution (5 ml) was serially diluted, plated onto brain heart infusion  agar plates, and then incubated under aerobic conditions at 37°C to determine the CFUs count , 16.For the determination of the dry weight and amount of water-insoluble/soluble EPSs and IPSs, the remaining solution (4.9 ml) was centrifuged (3000 ×g) for 20 min at 4°C. The biofilm pellet was resuspended and washed twice in the same volume of water. Mix the water-washed the biofilms pellet with 95% alcohol and put it in a refrigerator at -20°C for at least 18 h to precipitate the water-soluble extracellular polysaccharides (EPSs). Then calculate the content of water-soluble EPSs in the biofilms. The washed biofilms pellet was evenly divided into two portions, lyophilized, and weighed to determine the dry weight. One part used 1 N sodium hydroxide to extract water-insoluble EPSs from the dried precipitate. The other part was used to calculate the content of intracellular polysaccharides (IPSs), as detailed elsewhere .S. mutans biofilms by the CS exposure.The final pH values of the old culture media were also determined during the experimental period using a glass electrode  to investigate the change in acidogenicity of 3) divided by the substratum (hydroxyapatite surface) area (μm2). The three-dimensional architecture of the biofilms was visualized using ZEN 2.3 (blue edition) . The original confocal data was uploaded to ZEN 2.3 software and the intensity of green and red fluorescence in the full thickness of biofilms layers were captured automatically. The software reconstructed the 2-dimensional intensity of fluorescence in all the layers to a 3-dimensional volume stack [CLSM analysis was performed to confirm the results of microbiological and biochemical studies. To investigate the difference in bacterial cells, the 74-h-old biofilms were stained at room temperature in the dark for 30 min using the Film Tracer LIVE/DEAD Biofilm viability kit L10316 . The final concentrations of SYTO®9 and propidium iodide (PI) were 6.0 and 30 μM, respectively. This viability kit was based on plasma membrane integrity to determine live and dead cells. In this study, we regarded the cells with intact membranes (green) as live cells, whereas cells with damaged membranes (red) were regarded as dead cells. The excitation/emission wavelengths were 480/500nm for SYTO®9 and 490/635nm for PI for collecting the fluorescence. The stained live and dead bacterial cells were observed with an LSM 510 META microscope  equipped with argon-ion and helium–neon lasers. All confocal fluorescence images were taken with an EC Plan-Neofuar 10x/0.30 M27 objective lens. A stack of slices in 6.4 μm step sizes was captured from the top to the bottom of the biofilms. The biovolume and thickness of live and dead cells were quantified from the entire stack using COMSTAT image-processing software. The biovolume is defined as the volume of the biomass  to label the newly formed EPSs. As described above, the stained EPSs were observed with an LSM 510 META microscope  (objective: EC Plan Neofuar 10x/0.30 M27) equipped with argon-ion and helium-neon lasers and visualized using ZEN 2.3. A stack of slices in 7.8 μm step sizes was captured from the top to the bottom of the biofilms. Four independent experiments were performed, and five image stacks per experiment were collected. The EPSs biovolume and thickness were quantified from the confocal stacks using COMSTAT.The EPSs of 74-h-old biofilms were also investigated by simultaneous in situ labeling as described elsewhere . BrieflyS. mutans biofilms were calculated using the dry weight, which was derived from the biochemical study above, and the total biovolume of the biofilms (live cells + dead cells + EPSs), which was derived from the CLSM study above. The biofilm density (μg/μm3) is defined as the dry weight (μg/μm2) divided by the total biovolume of the biofilms (μm3/μm2) [The density of the 74-h-old μm3/μm2) .p-value was < 0.05.All experiments (except CLSM and SEM) were performed in duplicate, and at least six different experiments were conducted. The data are presented as mean ± standard deviation. Inter-group differences were estimated using a one-way analysis of variance, followed by a post hoc multiple comparisons (Tukey) test to compare multiple means  . Values were considered statistically significant when the S. mutans biofilms formation was inhibited by CS exposure. In the experimental results of microbial and biochemical studies, all CS exposure groups reduced the dry weight accumulation, water-insoluble/soluble EPSs, and IPSs formation of the 74-h-old S. mutans biofilms in a frequency-dependent manner, and the lowest values in the six times/day CS exposure group compared with the control group and other CS exposure groups (p < 0.05). There was no significant difference in bacterial activity (CFUs) counts among all CS exposure groups (p > 0.05).74-h-old S. mutans biofilms formation. Acidogenicity during S. mutans biofilms formation was affected by CS exposure. Although the acidogenicity of S. mutans biofilms before the CS exposure was not different from each other (p > 0.05) (0 ~ 21 h of biofilm formation). But the pH in the culture medium started to change after exposure with CS, the acidogenicity was inhibited at all of the CS exposure groups (p < 0.05) (21 ~ 74 h). The inhibition of acidogenicity of biofilms was enhanced with the increase of CS exposure frequency, and the highest inhibition ability was shown in the six times/day CS exposure group (p < 0.05). Furthermore, in all of the CS exposure periods and non-CS exposure periods (21 ~ 74 h), the pH values of the culture medium of S. mutans biofilms in the six times/day CS exposure group were significantly different from the control group (p < 0.05).S. mutans biofilms showed different results under different times of CS exposure (S. mutans biofilms (p < 0.05). In the percent of total biovolume, the proportion of live cell biovolume in each exposure group was between 60% and 70% . With the increase of CS exposure frequency, the anti-EPSs formation increased in a frequency-dependent manner. EPSs and bacterial microcolony images of 74-h-old S. mutans biofilms showed minimal concentrations of homogeneous structures of EPSs covering and surrounding bacterial microcolonies in the six times/day CS exposure group .The density of S. mutans biofilm model in vitro. S. mutans biofilm model is unable to precisely mimic the complex microbial community in dental biofilms, the mono-species biofilm is advantageous in examining the mechanisms of S. mutans in biofilms, in biofilms with sufficient performance to validate the data reproducibility and reduce variance [This study was to investigate the differences in growth, virulence (EPSs and acidogenicity), and viability of cariogenic biofilms according to the different times of CS exposure (simulate different smoking times each day) using an variance , 21, 22.S. mutans biofilm. The six times/day CS exposure group showed the lowest dry weight accumulation compared with the control group and other CS exposure groups (p < 0.05). CS inhibited the dry weight accumulation of S. mutans biofilm in a frequency-dependent manner. There was no significant difference in the bacterial activity between all CS exposure groups and the control group (p > 0.05). CLSM revealed that the live/dead cells bacterial biovolume, bacterial thickness, and total biovolume in 74-h-old S. mutans biofilms were frequency-dependently reduced by CS exposure (p < 0.05). The lowest values were shown in the six times/day CS exposure group. With the increase of CS exposure times, the number of live/dead cell microcolonies decreased and the arrangement of microcolonies was loose. However, the morphology of microcolonies did not change significantly, microcolonies in each treatment group that were similar to those in the control group could be observed . CS exposure inhibited the synthesis of polysaccharides of 74-h-old S. mutans biofilm in a frequency-dependent manner, which was consistent with the CLSM findings of the biovolume and thickness of EPSs (p < 0.05). Therefore, CS had an obvious damage effect on the EPSs formation of biofilms, and the decreased biovolume and thickness of EPSs may affect the growth height of bacterial cells .Acid production by carbohydrate and acid tolerance in a low pH environment is one of the main toxicity characteristics of . mutans , which a. mutans –27. In tS. mutans biofilms in a frequency-dependent manner in vitro, which was well supported by the measurements of biofilm dry weight , and total biovolume were detected in the six times/day CS exposure group. CLSM revealed that the thickness of bacteria (live or dead cells) was negatively correlated with the frequency of CS exposures, The lowest bacterial thickness detected in the six times/day CS exposure group also confirmed that the coverage of mixture chemical substances produced by cigarette combustion continuously covered on the surface of the S. mutans biofilms inhibited the increase in bacterial thickness , pH, and flow rates were of great significance in oral mucosal immunity, and salivary S-IgA antibodies generated by the mucosal immune system were an important role in the immune response against dental caries. Low concentrations of salivary sIgA were correlated with a higher prevalence of dental caries in smokers [Lactobacilli and S. mutans increase the susceptibility to dental caries [The results of this study support CS exposure in inhibiting l caries –13, whic smokers . It has  smokers . A decrel caries , which min vitro study supported the fact that CS exposure inhibited the growth of S. mutans biofilms because the mixture of chemical substances produced by cigarette combustion changed the colonizing environment on the surface of the hydroxyapatite disc, rather than sterilizing or killing cells. In this study, the frequency of CS exposure was the main variable, and there was no interference from other oral factors. Although only one cariogenic bacterium was included in this study, these findings provide a novel insight into clarifying the biological effects of CS on cariogenic biofilm growth.In general, the results S. mutans biofilm in vitro study, the anti-cariogenic biofilm formation was enhanced with a dose (frequency)-dependent at which frequency has more influence in the present findings.CS exposure inhibited the growth of


The studies, investigating the association of low-density lipoprotein cholesterol (LDL-C) with metabolic syndrome (MetS) are limited with controversial conclusions. Therefore, this study aimed at revealing the specific relationship between the serum LDL-C levels and MetS prevalence in a large working population.Secondary data analysis of a cross-sectional study, conducted between 2012 and 2016 in Spain on participants aged within the range of 20–70 years, involved 60,799 workers. Logistic regression analysis was applied to evaluate the association between the levels of serum LDL-C and MetS prevalence.P < 0.05).Among the 60,799 workers, the prevalence of MetS was 9.0%. The odds ratios  of MetS prevalence were 1.27 (1.16–1.39) and 1.53 (1.41–1.65) for the individuals with the LDL-C levels in lower (< 103.8 mg/dL) and upper (> 135.8 mg/dL) tertiles as compared to those with the LDL-C levels in middle tertile (103.8–135.8 mg/dL) in the studied population. Similarly, a U-shaped relationship was also observed in male cohort. The serum LDL-C levels associated with the lowest risk of current MetS were 113.6 mg/dL and 117.6 mg/dL in the overall studied population and male cohort, respectively. The female workers with the levels of LDL-C higher than 135.0 mg/dL had an increased prevalence of MetS  levels of LDL-C increased MetS prevalence in female workers. Metabolic syndrome (MetS) consists of a cluster of metabolic abnormalities, including abdominal obesity, insulin resistance, systemic hypertension, atherogenic dyslipidemia, and hyperglycemia –4. It han = 1000) aged 20–70 years [Dyslipidemia is a characteristic feature of MetS, and is used as a biomarker for its diagnosis . The hig70 years . However70 years . Given tTherefore, in order to explore the specific association between the serum LDL-C levels and prevalence of MetS, this study was conducted as a secondary analysis using a large sample of a working population.www.Datadryad.org). Romero-Saldaña et al. transferred the ownership of the original data to the Datadryad website, and this website (database) permits users to freely download raw data for secondary analysis , Data from: Validation of a non-invasive method for the early detection of metabolic syndrome: a diagnostic accuracy test in a working population, Dryad, Dataset, 10.5061/dryad.cb51t54). A total of 60,799 participants , belonging to different socioeconomic sectors  were voluntarily recruited from Balearic Islands, Spain during the periodic assessment of their health, using random sampling between 2012 and 2016 [This study was conducted as a secondary analysis of data from a cross-sectional study . The useand 2016 . In the and 2016 . As statand 2016 .The variables investigated in this study included baseline characteristics, anthropometric measurements, and laboratory tests. All the anthropometric parameters were measured in triplicates by well-trained staff, and then their average was recorded. A 12-h overnight fasting venous blood sample was used for laboratory tests. The laboratory tests followed the standard procedures for clinical biochemical laboratories, as described previously . The folt-test or Pearson’s chi-squared test was used for the comparison between the two groups (MetS group and non-MetS group). In the present study, the serum LDL-C tertiles were used for the analysis based on a U-shaped relationship, which was observed between the levels of LDL-C and MetS prevalence. Multicollinearity test was performed. The following criteria were used for the selection of covariables for adjustment: introduction covariables in the basic model or removal of covariables from the full model led to a change in the regression coefficient (β) of LDL-C > 10%; the regression coefficient of covariables on dependent variable (current MetS) yielded a P-value < 0.1; or the covariables needed to be adjusted by clinical observations [P-value of < 0.05 was considered statistically significant.The data was expressed as number (percentage) or mean ± standard deviation according to characteristic variables . Unpaired rvations . A multirvations . The staP < 0.05). In addition, both the male and female participants with MetS had higher levels of serum LDL-C than those without MetS (P < 0.05).In this study, the prevalence of MetS was 9.0%  in the studied population. Among 34,827 male and 25,972 female participants, the prevalence of MetS was 11.8% (4097/34827) and 5.4% (1390/25872), respectively, based on the diagnostic criteria of MetS suggested by the NCEP-ATP III. Both the male and female participants, who developed MetS, were older than those without MetS  were screened out. Except the age judged by clinical significance, the other three variables were excluded. In addition, the covariates of gender, BMI, DBP, ABSI, and smoking met the criteria needed to be adjusted in the multivariate regression analysis. A U-shaped relationship was observed between the serum LDL-C levels and MetS prevalence in the overall studied population Fig. A. FurtheP < 0.05). The inflection point of serum LDL-C in male cohort was detected at the concentration of 117.6 mg/dL, above which no statistically significant difference was observed between increased LDL-C level and MetS prevalence as compared to those with the LDL-C levels below the inflection point   in the overall population  increased MetS prevalence. These findings might have implications in the management of serum LDL-C in subjects with MetS during clinical practice.n = 1000) aged within the range of 20–70 years, reported that there was no significant correlation between the serum LDL-C levels and MetS prevalence [n = 3871) and longitudinal (n = 2558) health screening study conducted in Japanese people, Oda pointed out that the LDL-C levels were related to MetS and can serve as a predictor for its development [Several previous studies tried to reveal the association between serum LDL-C and MetS but the conclusion are controversial. A cross-sectional study by Hajian-Tilaki et al., conducted on the representative samples of Iranian adults  would also show this pattern of relationship with LDL-C. Thirdly, since this study was just a secondary analysis based on a cross-sectional study, it could not deduce causality between the LDL-C level and current MetS statuses. In addition, the lack of several variables  in the raw data limited a further subgroup analysis.Both the low and high levels of serum LDL-C were observed to be related to the increasing prevalence of MetS both in the working population and male workers. However, only the high LDL-C levels were shown to be positively associated with an increase in the prevalence of MetS in female workers. These findings revealed that the management of LDL-C should be carried out taking into consideration the gender-specific differences in MetS. These findings also indicated that, in order to reduce the prevalence of MetS, it might be appropriate to pursue the lower levels of LDL-C among female population rather than male population.


Objectives: The present study aimed to assess the expression of caspase-1 and caspase-1-dependent processing of cytokines, such as interleukin (IL)-1β and IL-18, in the middle ear effusion of children with otitis media with effusion (OME) in order to identify the potential role of inflammasomes in OME.Methods: This study included 29 children scheduled for myringotomy with the insertion of tympanostomy tubes due to OME. Middle ear effusion (MEE) was collected during the surgery. Caspase-1, IL-1β, and IL-18 were assayed using enzyme-linked immunosorbent assay kits. The levels were compared between those with mucoid and serous MEE and those with and without a history of ventilation tube insertion.Results: Caspase-1, IL-1β, and IL-18 were detected in all samples. The caspase-1, IL-1β, and IL-18 levels did not significantly differ between mucoid samples and serous samples. No statistical significances were discovered in caspase-1, IL-1β, and IL-18 levels between with and without a history of ventilation tube groups. There was a significant negative correlation between IL-1β and IL-18 and the duration of OME (p < 0.05). However, no significant correlation was found between caspase-1 and disease duration.Conclusions: Inflammasomes may participate in the inflammatory process of OME. IL-1β and IL-18 levels in the MEE decreased over time. Otitis media with effusion (OME), a common disease in children, is characterized by the collection of fluid in the middle ear without acute inflammatory manifestations , 2. ThisInflammasomes are large protein complexes best known for their ability to control activation of pro-caspase-1, and active caspase-1 in turn processes pro-IL-1β and pro-IL-18 into their mature active forms, IL-1β and IL-18, respectively . InapproTherefore, we aimed to evaluate the expression levels of caspase-1, IL-1β, and IL-18 in the middle ear effusion (MEE) of children with OME in order to explore their clinical significance. To the best of our knowledge, we present the first study concerning MEE Caspase-1 and IL-18 levels in children with OME.This cross sectional study included children who underwent myringotomy with tube placement for OME our hospital between July 2020 and January 2021. Based on the limited reference , a confiDemographic and clinical data, including patient age, sex, medical history, auditory testing results, and type of MEE (mucoid or serous) were recorded. Informed consent was obtained from the parents of each patient for the experimental use of specimens. Approval for this study was granted by the Ethics Review Committee of our hospital.Prior to surgery, the external auditory canal was washed with 70% alcohol, and a radial incision was made in the anterior inferior quadrant of the tympanic membrane. MEE was collected directly into a 1 mL syringe with the help of a suction device. Each sample was classified as a mucoid or serous MEE based on its appearance and fluidity. Mucoid MEE was a thick and viscous effusion that nearly did not flow when the tube was inverted, while serous MEE was a thin and watery effusion that flowed immediately when the tube was inverted. We found bilateral effusions in one patient who was equally classified. The effusion samples were then transferred to Eppendorf tubes, and bilateral effusions were combined into one tube per patient, and stored at −80°C until analysis.After thawing, sterile saline was added to the samples according to their volume and viscosity, and the samples were then centrifuged at 1300 × g for 10 min. Supernatants from centrifuged samples were collected and diluted to 1:20. Effusions grossly contaminated with blood were excluded from the study. The total protein was measured in each sample using the Bradford Protein Assay Kit  following the manufacturer's instructions. The levels of caspase-1 , IL-1β , IL-18  were detected using enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.p < 0.05.Analyses were conducted using SPSS software . The Kolmogorov-Smirnov test was used to assess the normality of the data distribution. The results are expressed as medians and ranges. Statistical differences in the numerical data were analyzed using the Mann-Whitney U-test. The chi-square test was used to analyze categorical data and Linear regression was applied to adjust for covariate. Spearman's correlation was performed to determine correlations. Statistical significance was set at p > 0.05)  were included in this study. The median age was 55 months , the median disease duration was 12 months , and 8 (27.6%) patients had undergone myringotomy with tube placement before enrollment. The 29 patients in this study were divided into serous and mucoid groups. There were no significant differences between the two groups according to age, sex, duration of OME, and previous ventilation tube insertions ( > 0.05) . The medp > 0.05). The caspase-1, IL-1β, and IL-18 levels did not significantly differ between mucoid and serous MEE . Between the two groups, the distribution of sex and MEE type did not significantly differ . However, the children in group A were significantly younger  than those in group B . The median disease duration was significantly shorter in group A  than in group B  (p = 0.011). The difference in disease duration between the two groups could be due to the fact that patients who underwent multiple ventilation tube insertions tended to experience longer courses of OME. To adjust for age, linear regression was further performed. As a result, the caspase-1 and IL-18 levels did not significantly differ between groups (Previous studies have suggested that children who require multiple ventilation tube insertions may have MEE with different inflammatory composition . In thisn groups .p < 0.05)  . BecauseThe canonical inflammasome complex contains a nucleotide-binding domain and leucine-rich-repeat-containing [NLR] protein or an AIM2-like receptor [ALR] protein, apoptosis-associated speck-like protein containing a CARD [ASC], and pro-caspase-1 . RegardiOne study regarding the effects of IL-1β on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) in human middle ear cells suggested that IL-1β upregulates NKCC1 in middle ear epithelial cells, which is one of the important underlying mechanisms of excess fluid collection in OME . IL-1β cIt seems that mucoid and serous MEE have different compositions and biological signatures . RemodelIL-1β and IL-18 both belong to the proinflammatory IL-1 family . They arEarly studies suggested that the concentrations of tumor necrosis factor-α and C3 were higher in MEE samples from children who had undergone multiple ventilation tube insertions , 28. In This is the first study to evaluate the level of caspase-1 in MEE samples from children with OME and investigate the correlation between caspase-1 and clinical parameters. The results of this study indicate that inflammasomes may participate in the pathogenesis of OME. This study had several limitations. First, the collection of mucoid MEE was difficult, and samples were easily contaminated by blood during collection; therefore, the sample size was small. Second, there was no effusion in a healthy middle ear cavity; hence, we did not set up a control group. Third, we only measured the level of protein and lacked the measurement of mRNA. We recommend further studies with larger sample sizes and diverse detection methods to confirm the validity of our findings.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Ethics Review Committee of Beijing Childern's Hospital. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.SLiu, JZ, and XN: conceptualization. SLiu, LG, YL, XW, and SLi: data curation. SLiu and LG: formal analysis. JZ and XN: funding acquisition and writing–review and editing. MC, WL, JZ, and XN: methodology. SLiu: project administration, visualization, and writing–original draft. All authors contributed to the article and approved the submitted version.This work was supported by Pediatric Medical Coordinated Development Center of Beijing Hospitals Authority (XTYB201828) and Beijing Hospitals Authority′ Ascent Plan (DFL20191201).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.